Evolutionary diversity of bile salts in reptiles and mammals, including analysis of ancient human and extinct giant ground sloth coprolites

Abstract

Background: Bile salts are the major end-metabolites of cholesterol and are also important in lipid and protein digestion and in influencing the intestinal microflora. We greatly extend prior surveys of bile salt diversity in both reptiles and mammals, including analysis of 8,000 year old human coprolites and coprolites from the extinct Shasta ground sloth (Nothrotherium shastense).

Results: While there is significant variation of bile salts across species, bile salt profiles are generally stable within families and often within orders of reptiles and mammals, and do not directly correlate with differences in diet. The variation of bile salts generally accords with current molecular phylogenies of reptiles and mammals, including more recent groupings of squamate reptiles. For mammals, the most unusual finding was that the Paenungulates (elephants, manatees, and the rock hyrax) have a very different bile salt profile from the Rufous sengi and South American aardvark, two other mammals classified with Paenungulates in the cohort Afrotheria in molecular phylogenies. Analyses of the approximately 8,000 year old human coprolites yielded a bile salt profile very similar to that found in modern human feces. Analysis of the Shasta ground sloth coprolites (approximately 12,000 years old) showed the predominant presence of glycine-conjugated bile acids, similar to analyses of bile and feces of living sloths, in addition to a complex mixture of plant sterols and stanols expected from an herbivorous diet.

Conclusions: The bile salt synthetic pathway has become longer and more complex throughout vertebrate evolution, with some bile salt modifications only found within single groups such as marsupials. Analysis of the evolution of bile salt structures in different species provides a potentially rich model system for the evolution of a complex biochemical pathway in vertebrates. Our results also demonstrate the stability of bile salts in coprolites preserved in arid climates, suggesting that bile salt analysis may have utility in selected paleontological research.

Figure 1

Representative bile salts and their structures. (A) Simplified version of the bile salt synthetic pathway showing the three major classes of compounds that can serve as primary bile salts (C₂₇ bile alcohols, C₂₇ bile acids, and C₂₄ bile acids). (B) All bile salts are derived from cholesterol (topmost structure), illustrated with the carbon atoms numbered and the steroid rings labelled A, B, C, and D. Iguanian lizards utilize 5α C₂₄ bile acids such as 5α-cholic acid (allocholic acid) that have an A/B ring juncture that is trans, resulting in an overall planar and extended structure of the steroid rings (see representation of A, B, and C rings on the right side). Crocodylians utilize trihydroxy 5β C₂₇ bile acids with hydroxylation at C-12 in addition to the default 3α, 7α-dihydroxylation. One of the two most common primary salts in mammals is chenodeoxycholic acid (CDCA), the stem C₂₄ bile acid that has the default default 3α, 7α-dihydroxylation.

Figure 2

Bile salt synthetic pathways. The enzymatic pathways involved in bile salt synthesis have so far been elucidated only in humans and rodents. The pathway shown is the 'neutral' pathway, as opposed to the 'acidic' pathway that modifies the side-chain before the steroid nucleus [6,7]. The ultimate production of the default C₂₄ bile acid chenodeoxycholic acid (CDCA) requires multiple changes to the original cholesterol structure including 7α-hydroxylation, isomerization of the C-5 - C-6 double bond (from cholesterol) to form a Δ⁴ compound coupled with oxidation of the 3β-hydroxy group to a 3-oxo group, stereospecific reduction of the 3-oxo group to a 3α-hydroxy group, hydroxylation of the terminal carbon atom of the side-chain, oxidation of the side-chain (to form a C₂₇ bile acid), and shortening of the side-chain. Animals using C₂₇ bile alcohols or C₂₇ bile acids as their primary bile salts presumably require fewer enzymatic steps than those synthesizing C₂₄ bile acids. The enzymes involved in 5α-reduction leading to 5α-bile salts are currently unknown. The stem C₂₇ 5β-bile alcohol would be 3α,7α,27-trihydroxy-5β-cholestan, while the stem C₂₇ 5β-bile acid would be 3α,7α-dihydroxy-5β-cholestan-27-oic acid. Note that some steps in the pathway require more than one enzyme (e.g., isomerization of the 3β-hydroxy group of cholesterol to the 3α configuration), whereas some enzymes (e.g., CYP27A1) can catalyze more than one reaction. Double bonds or additional hydroxyl groups may be added to the nucleus or side-chain, either on an intermediate or on the completed stem bile alcohol or acid.

Figure 3

Bile salts of Squamata. The bile salt variation of lizards is overlaid on a tree that represents a summary of current knowledge of squamate phylogeny based on molecular data. Groups of squamates are color-coded following the nomenclature in the review by Hedges and Vidal [18]. Major bile salts are those constituting more than 50% of total biliary bile salts. Minor bile salts account for less than 50% but more than 10% of total bile salts. The bile salts of Iguania (group A) are dominated by 5α (allo) bile acids. The C₂₇ bile acids of species within Anguimorpha (group B) are almost entirely varanic acid. The bile salts of Laterata (group C) are mainly CA with minor fractions of alloCA in some species. The bile salts of species sampled within Scinciformata showed the most diversity of any of the five squamate groups surveyed, with some species having mainly varanic acid (C₂₇ bile acid) and others having mainly alloCA. Species within Gekkota (group E) had mainly CA and CDCA as major bile salts. Abbreviations: alloCA, allo (5α)-cholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid.

Figure 4

Bile salts of snakes. The bile salt variation of snakes is overlaid on a tree that represents a summary of current knowledge of snake phylogeny based on molecular data. Major bile salts are those constituting more than 50% of total biliary bile salts. Minor bile salts account for less than 50% but more than 10% of total bile salts. Abbreviations: CA, cholic acid; 23R-OH, 23R-hydroxylated C₂₄ bile acids (mainly 23R-hydroxylated cholic acid); Δ22, C₂₄ bile acids with double bond at C22-23; C₂₃, C₂₃ bile acids; pythoCA, pythocholic acid.

Figure 5

Bile salts of mammals. The bile salt variation of mammals is overlaid on a mammalian phylogeny based on molecular data [23], with the single revision to place Afrotheria and Xenarthra as sister groups. In the "Other C₂₄ acids" columns are included the 1α-, 1β-, and 15α-hydroxylated bile acids found in some marsupials and the 6α- and 6β-hydroxylated bile acids of rodents and Suidae, as well as deoxycholic acid found in various species. Abbreviations: CDCA, chenodeoxycholic acid; CA, cholic acid; DCA, deoxycholic acid.

Figure 6

Overall bile salt variation in reptiles, birds, and mammals. The phylogeny depicted has Testudines as sister-group to Aves/Crocodylia. Lizards are paraphyletic but are indicated here as a single group for comparison purposes. Based on the patterns of bile salt variation across vertebrates, the ancestral bile salt profiles at nodes A, B, and C can be inferred by parsimony. The ancestral phenotype at node A would likely be C₂₇ bile alcohols, the phenotype found in all living lobe-finned, jawless, and cartilaginous fish surveyed to date. The ancestral phenotype at node B would likely be C₂₇ bile alcohols and trihydroxy-C₂₇ bile acids, the major bile salts of Crocodylia and paleognath birds. The most recent common ancestor to tuatara, lizards, and snakes (node C) would additionally have the ability to produce 24R-hydroxylated C₂₇ bile acids, the main bile salts of tuatara and lizards within Anguimorpha and Scinciformata. Based on the phylogeny depicted, there appear to be several 'innovations' unique to certain groups: (1) 24R-hydroxylation of C₂₇ bile acids by lepidosaurs as mentioned above, (2) 22-hydroxylation of C₂₇ bile acids by Testudines, and (3) production of 7-deoxy and C₂₃ bile acids by snakes. The figure does not show all the known bile salt modifications, but only some of the more common ones within in each group.

Figure 7

Overall bile salt variation across vertebrates. Variation of bile salts is overlaid on a vertebrate phylogeny, with turtles placed as sister group to birds/crocodiles, frogs and salamanders as sister groups, and placental mammals and marsupials as sister groups [37]. Lizards are paraphyletic but are indicated here as a single group for comparison purposes. Unresolved relationships are depicted as polyotomies. The figure shows two "shifts to the right" from 5α-C₂₇ bile alcohols to C₂₄ bile acids as the bile salt synthetic pathway presumably grew in length: (1) from Agnatha to ray-finned fish and (2) from lobe-finned fish to tetrapods. Note that C₂₇ bile acids are common in reptiles and amphibians but uncommon in fish. Within amphibians, there is only preliminary data on caecilians based on analysis of two specimens, both of which showed only bile alcohols but with the orientation of the 5-hydroxyl group as yet undetermined pending additional analyses (LR Hagey, unpublished data).

Figure 8

Illustration of 5α bile salts. The 5α series of C₂₇ bile alcohol, C₂₇ bile acid, and C₂₄ bile acid structures are illustrated with examples of animals (if any) whose major bile salts are in these classes. The structures are the stem structures for each of the three major classes of bile salts. Multiple enzymes are involved in the transition indicated by arrow A. The enzymes responsible for 5α-reduction of bile salt precursors are currently unknown. If 5α-bile salt pathways are homologous to 5β pathways, the reaction indicated by arrow B would be carried out by CYP27A1. Side-chain cleavage (arrow C) is likely a peroxisomal reaction in most or all animals. Arrow D covers all the possible additional modifications to stem bile salt structures (e.g., additional hydroxylation of the nucleus or side-chain, introduction of oxo groups, unsaturation of the side-chain, etc.).

Figure 9

Illustration of 5β bile salts. The 5β series of C₂₇ bile alcohol, C₂₇ bile acid, and C₂₄ bile acid structures are illustrated with examples of animals (if any) whose major bile salts are in these classes. The structures are the stem structures for each of the three major classes of bile salts. Multiple enzymes are involved in the transition indicated by arrow A. Arrow B is carried out by CYP27A1 in humans and rodents. Side-chain cleavage (arrow C) is likely a peroxisomal reaction in most or all animals. Arrow D covers all the possible additional modifications to stem bile salt structures (e.g., additional hydroxylation of the nucleus or side-chain, introduction of oxo groups, unsaturation of the side-chain, etc.).